Low molecular weight thyroid stimulating hormone receptor (TSHR) agonists

ABSTRACT

Disclosed are oxo-hydroquinazolines that are useful as selective TSHR agonists. The compounds may be used for detecting or treating thyroid cancer, or treating a bone degenerative disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 13/125,045, filedApr. 19, 2011, which is the U.S. National Stage of InternationalApplication No. PCT/US2008/011958, filed Oct. 20, 2008, published inEnglish under PCT Article 21(2), both of which are herein incorporatedby reference in their entirety.

FIELD

Disclosed herein are compounds that are thyroid stimulating hormonereceptor (TSHR) agonists for diagnostic, analytical and therapeuticpurposes.

BACKGROUND

Thyroid-stimulating hormone (TSH) is a heterodimeric glycoproteinhormone that regulates thyroid homeostasis. TSH is involved in thegrowth and function of thyroid follicular cells. Cellular responses toTSH are mediated via the TSH receptor (TSHR) which is a distinct seventransmembrane-spanning receptor. TSHR is the major regulator of thyroidgland (and most thyroid cancer) function and is expressed in bone andadipocytes (fat) precursor cells. Activation of TSHR by its endogenoushormone TSH is required for normal thyroid homeostasis but may alsoregulate bone and fat biology.

The thyroid gland is, as is well known, one site of metabolic controlwithin the body. Cancer of the thyroid gland is not particularly common,but the high rate of disease re-occurrence necessitates long termsurveillance. Usually, during treatment for cancer of the thyroid, themajority of the thyroid tumor is removed, but a small amount oftenremains that must be treated by radioactive iodide therapy. Indeed,thyroid cancer is characterized by a high likelihood of relapses in upto 30% of patient, even after successful therapy. Therefore, follow-upscreening is necessary.

Following surgery, it is necessary to treat the patient with thyroidhormone, as the patient will no longer produce this. One role of thethyroid gland is to take up iodine from the body. Hence, it should bepossible to treat any remaining tumor cells with radioactive iodide.Unfortunately, though, thyroid cancer cells do not take up iodine well.So, in order for the radioactive iodine to work, the patient has toeither be treated with recombinant TSH or have thyroid hormone treatmentwithdrawn in order to elevate natural TSH levels, to stimulate iodideuptake. Withdrawal of thyroid hormone has quite unpleasant side effectsfor the patient, particularly fatigue, muscle cramps, puffiness andconstipation. Thus, at the present time, recombinant human TSH (rhTSH,Thyrogen®, Genzyme) is used clinically for screening after surgery inpatients with well-differentiated thyroid cancer. However, rhTSH is adimeric glycoprotein molecule that is made by genetic engineering inhuman cells. It is difficult to produce, requires stringent qualitycontrol and must be administered parenterally.

SUMMARY

Disclosed herein are compounds having formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹-R⁴ are each independently H, hydroxyl, alkyl, alkoxy, aminocarbonyl,or halogen;

R⁵ is H, alkyl, aryl, aralkyl, or aminocarbonyl;

A represents —N═C(R¹⁵)— (wherein a bond at the left end bonds to thebenzene ring of formula I above and a bond at the right end bonds to thenitrogen heteroatom of formula I above) or —NH—CH(R¹⁵)— (wherein a bondat the left end bonds to the benzene ring of formula I above and a bondat the right end bonds to the nitrogen heteroatom of formula I above);

R¹⁵ is represented by formula II:

wherein:

R⁶-R⁹ are each independently H, hydroxyl, alkyl, or alkoxy;

R¹⁰-R¹⁴ are each independently H, hydroxyl, alkyl, alkoxy, oraminocarbonyl; and

X is O, S or N(H);

with the proviso that the compound of formula I is not

Also disclosed are additional Compounds of formula VI:

wherein R²⁰ is aryl or heteroaryl; R²¹ is aryl or heteroaryl; R²² is R¹⁵of formula II as shown in claim in claim 1, heteroaryl, or aryl.

Also disclosed herein is a method for detecting thyroid cancer in asubject, comprising administering to the subject a diagnosticallyeffective amount of at least one compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹-R⁴ are each independently H, hydroxyl, alkyl, alkoxy, aminocarbonyl,or halogen;

R⁵ is H, alkyl, aryl, aralkyl, or aminocarbonyl;

A represents —N═C(R¹⁵)— (wherein a bond at the left end bonds to thebenzene ring of formula I above and a bond at the right end bonds to thenitrogen heteroatom of formula I above) or —NH—CH(R¹⁵)— (wherein a bondat the left end bonds to the benzene ring of formula I above and a bondat the right end bonds to the nitrogen heteroatom of formula I above);

R¹⁵ is represented by formula II:

wherein:

R⁶-R⁹ are each independently H, hydroxyl, alkyl, or alkoxy;

R¹⁰-R¹⁴ are each independently H, hydroxyl, alkyl, alkoxy, oraminocarbonyl; and

X is O, S or N(H);

or at least one compound of formula VI.

A further embodiment disclosed herein involves a method for treating orpreventing a bone degenerative disorder comprising administering atherapeutically effective amount of at least one compound of formula Ior at least one compound of formula VI.

Also disclosed herein is a method for treating thyroid cancer in asubject, comprising administering to the subject a therapeuticallyeffective amount of at least one compound of formula I or at least onecompound of formula VI.

An additional embodiment disclosed herein involves a method foractivating a thyroid stimulating hormone receptor in an assay,comprising contacting the thyroid stimulating hormone receptor with atleast one compound of formula I or at least one compound of formula VI.

Also disclosed herein are pharmaceutical compositions comprising apharmaceutically acceptable carrier and at least one compound of formulaI or at least one compound of formula VI.

The foregoing and other objects, features, and advantages will becomemore apparent from the following detailed description, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graph depicting data showing that a compound disclosed hereinis a full and selective agonist.

FIG. 2 is a graph depicting data demonstrating the potency of severalcompounds disclosed herein.

FIG. 3 is a graph of data for the agonist activity of several compoundsdisclosed herein in primary cultures of human thyrocytes.

FIG. 4 is a graph of data of in vivo activity of a compound disclosedherein in a mouse model.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.

Variables such as R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, n, X and Y, usedthroughout the disclosure are the same variables as previously definedunless stated to the contrary.

“Administration of” and “administering a” compound should be understoodto mean providing a compound, a prodrug of a compound, or apharmaceutical composition as described herein. The compound orcomposition can be administered by another person to the subject (e.g.,intravenously) or it can be self-administered by the subject (e.g.,tablets).

“Optional” or “optionally” means that the subsequently described eventor circumstance can but does not need to occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not.

“Derivative” refers to a compound or portion of a compound that isderived from or is theoretically derivable from a parent compound.

The term “subject” includes both human and veterinary subjects.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” refers to inhibitingthe full development of a disease or condition, for example, in asubject who is at risk for a disease such as a hormone receptor mediateddisorder, particularly a thyroid disorder, such as a hyperthyroid orhypothyroid disorder. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology. By the term “coadminister” is meant that each ofat least two compounds be administered during a time frame wherein therespective periods of biological activity overlap. Thus, the termincludes sequential as well as coextensive administration of two or moredrug compounds.

The terms “pharmaceutically acceptable salt” or “pharmacologicallyacceptable salt” refers to salts prepared by conventional means thatinclude basic salts of inorganic and organic acids, including but notlimited to hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid,acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid,fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid,phenylacetic acid, mandelic acid and the like. “Pharmaceuticallyacceptable salts” of the presently disclosed compounds also includethose formed from cations such as sodium, potassium, aluminum, calcium,lithium, magnesium, zinc, and from bases such as ammonia,ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine,choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine,procaine, N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.These salts may be prepared by standard procedures, for example byreacting the free acid with a suitable organic or inorganic base. Anychemical compound recited in this specification may alternatively beadministered as a pharmaceutically acceptable salt thereof.“Pharmaceutically acceptable salts” are also inclusive of the free acid,base, and zwitterionic forms. Descriptions of suitable pharmaceuticallyacceptable salts can be found in Handbook of Pharmaceutical Salts,Properties, Selection and Use, Wiley VCH (2002). When compoundsdisclosed herein include an acidic function such as a carboxy group,then suitable pharmaceutically acceptable cation pairs for the carboxygroup are well known to those skilled in the art and include alkaline,alkaline earth, ammonium, quaternary ammonium cations and the like. Suchsalts are known to those of skill in the art. For additional examples of“pharmacologically acceptable salts,” see Berge et al., J. Pharm. Sci.66:1 (1977).

“Saturated or unsaturated” includes substituents saturated withhydrogens, substituents completely unsaturated with hydrogens andsubstituents partially saturated with hydrogens.

The term “acyl” refers group of the formula RC(O)— wherein R is anorganic group.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A“lower alkyl” group is a saturated branched or unbranched hydrocarbonhaving from 1 to 10 carbon atoms. Preferred alkyl groups have 1 to 4carbon atoms. Alkyl groups may be “substituted alkyls” wherein one ormore hydrogen atoms are substituted with a substituent such as halogen,cycloalkyl, alkoxy, amino, hydroxyl, aryl, or carboxyl.

The term “alkenyl” refers to a hydrocarbon group of 2 to 24 carbon atomsand structural formula containing at least one carbon-carbon doublebond.

The term “alkynyl” refers to a hydrocarbon group of 2 to 24 carbon atomsand a structural formula containing at least one carbon-carbon triplebond.

The terms “halogenated alkyl” or “haloalkyl group” refer to an alkylgroup as defined above with one or more hydrogen atoms present on thesegroups substituted with a halogen (F, Cl, Br, I).

The term “cycloalkyl” refers to a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. The term “heterocycloalkyl group” is acycloalkyl group as defined above where at least one of the carbon atomsof the ring is substituted with a heteroatom such as, but not limitedto, nitrogen, oxygen, sulfur, or phosphorous.

The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl,halogenated alkyl and cycloalkyl groups as described above. A “loweraliphatic” group is a branched or unbranched aliphatic group having from1 to 10 carbon atoms.

The term “alkoxy” refers to a straight, branched or cyclic hydrocarbonconfiguration and combinations thereof, including from 1 to 20 carbonatoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to 4carbon atoms, that include an oxygen atom at the point of attachment. Anexample of an “alkoxy group” is represented by the formula —OR, where Rcan be an alkyl group, optionally substituted with an alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupas described above. Suitable alkoxy groups include methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxycclopropoxy, cyclohexyloxy, and the like.

“Alkoxycarbonyl” refers to an alkoxy substituted carbonyl radical,—C(O)OR, wherein R represents an optionally substituted alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety.

The term “alkyl amino” refers to alkyl groups as defined above where atleast one hydrogen atom is replaced with an amino group.

“Aminocarbonyl” alone or in combination, means an amino substitutedcarbonyl (carbamoyl) radical, wherein the amino radical may optionallybe mono- or di-substituted, such as with alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyland the like. An aminocarbonyl group may be —N(R)—C(O)—R (wherein R is asubstituted group or H) or —C(O)—N(R). An “aminocarbonyl” is inclusiveof an amido group. A suitable aminocarbonyl group is acetamido.

The term “aryl” refers to any carbon-based aromatic group including, butnot limited to, benzene, naphthalene, etc. The term “aromatic” alsoincludes “heteroaryl group,” which is defined as an aromatic group thathas at least one heteroatom incorporated within the ring of the aromaticgroup. Examples of heteroatoms include, but are not limited to,nitrogen, oxygen, sulfur, and phosphorous. The aryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone,aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can beunsubstituted.

“Carbonyl” refers to a radical of the formula —C(O)—.Carbonyl-containing groups include any substituent containing acarbon-oxygen double bond (C═O), including acyl groups, amides, carboxygroups, esters, ureas, carbamates, carbonates and ketones and aldehydes,such as substituents based on —COR or —RCHO where R is an aliphatic,heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary,or quaternary amine.

“Carboxyl” refers to a —COOH radical. Substituted carboxyl refers to—COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or acarboxylic acid or ester.

The term “hydroxyl” is represented by the formula —OH.

The term “hydroxyalkyl” refers to an alkyl group that has at least onehydrogen atom substituted with a hydroxyl group. The term “alkoxyalkylgroup” is defined as an alkyl group that has at least one hydrogen atomsubstituted with an alkoxy group described above.

The term “amine” or “amino” refers to a group of the formula —NRR′,where R and R′ can be, independently, hydrogen or an alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above.

The term “amide” or “amido” is represented by the formula —C(O)NRR′,where R and R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above. A suitable amido group is acetamido.

The term “aralkyl” refers to an aryl group having an alkyl group, asdefined above, attached to the aryl group, as defined above. An exampleof an aralkyl group is a benzyl group.

Optionally substituted groups, such as “optionally substituted alkyl,”refers to groups, such as an alkyl group, that when substituted, havefrom 1-5 substituents, typically 1, 2 or 3 substituents, selected fromalkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy, amino,aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, halogen, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, hydroxy,sulfonyl, thiol and thioalkoxy. In particular, optionally substitutedalkyl groups include, by way of example, haloalkyl groups, such asfluoroalkyl groups, including, without limitation, trifluoromethylgroups.

A “therapeutically effective amount” or “diagnostically effectiveamount” refers to a quantity of a specified agent sufficient to achievea desired effect in a subject being treated with that agent. Forexample, this may be the amount of a compound disclosed herein useful indetecting or treating thyroid cancer in a subject. Ideally, atherapeutically effective amount or diagnostically effective amount ofan agent is an amount sufficient to inhibit or treat the disease withoutcausing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount or diagnostically effective amount ofan agent will be dependent on the subject being treated, the severity ofthe affliction, and the manner of administration of the therapeuticcomposition.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The suitability and techniques involved in makingand using prodrugs are well known by those skilled in the art. For ageneral discussion of prodrugs involving esters see Svensson and TunekDrug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs,Elsevier (1985).

Pharmaceutically acceptable prodrugs refer to compounds that aremetabolized, for example, hydrolyzed or oxidized, in the subject to forman antiviral compound of the present disclosure. Typical examples ofprodrugs include compounds that have one or more biologically labileprotecting groups on or otherwise blocking a functional moiety of theactive compound. Prodrugs include compounds that can be oxidized,reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed,dehydrolyzed, alkylated, dealkylated, acylated, deacylated,phosphorylated, dephosphorylated to produce the active compound. Ingeneral the prodrug compounds disclosed herein possess hormone receptormodulating activity and/or are metabolized or otherwise processed invivo to form a compound that exhibits such activity.

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compound also are contemplated. Avariety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions which willnot affect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

When an amine is deprotected, the resulting salt can readily beneutralized to yield the free amine. Similarly, when an acid moiety,such as a phosphonic acid moiety is unveiled, the compound may beisolated as the acid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one ormore asymmetric centers; thus these compounds can exist in differentstereoisomeric forms. Accordingly, compounds and compositions may beprovided as individual pure enantiomers or as stereoisomeric mixtures,including racemic mixtures. In certain embodiments the compoundsdisclosed herein are synthesized in or are purified to be insubstantially enantiopure form, such as in a 90% enantiomeric excess, a95% enantiomeric excess, a 97% enantiomeric excess or even in greaterthan a 99% enantiomeric excess, such as in enantiopure form.

It is understood that substituents and substitution patterns of thecompounds described herein can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art and further by themethods set forth in this disclosure. Reference will now be made indetail to the presently preferred compounds.

I. Compounds

Disclosed herein are low molecular weight (for example, less than 1000daltons) compounds that activate TSHR. These compounds may be orallyadministered. Data is presented below demonstrating the efficacy of thecompounds in cells expressing human TSHRs in culture. Certain compoundsdisclosed herein are selective agonists for TSHR (i.e, the compounds donot activate or modulate other hormone receptors, particularly LHCGR andFSHR). The compounds also may be full agonists for TSHR.

The TSHR agonists disclosed herein enhance or activate a TSH signalingpathway. The TSHR agonists may stimulate the TSHR-mediated signaling bythemselves, or stimulate TSHR-mediated signaling by enhancing thebiological activity of endogenous TSH or another administered (i.e.,exogenous) TSHR agonist. Although not bound by any theory, it isbelieved that in certain embodiments the TSHR agonists disclosed hereinspecifically bind TSHR (in particular, the transmembrane domain of TSHR)which then transduces TSHR-mediated intracellular signaling inthyrotrophs or other cells naturally expressing TSHR or cells modifiedto express TSHR. The term “specific binding” and its cognates refer toan interaction with an affinity constant K_(A) of less than 100micromolar, particular 100 nanomolar, and preferably less than 50nanomolar.

Examples of the compounds are represented by formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹-R⁴ are each independently H, hydroxyl, alkyl, alkoxy, aminocarbonyl,or halogen;

R⁵ is H, alkyl, aryl, aralkyl, or aminocarbonyl;

A represents —N═C(R¹⁵)— (wherein a bond at the left end bonds to thebenzene ring of formula I above and a bond at the right end bonds to thenitrogen heteroatom of formula I above) or —NH—CH(R¹⁵)— (wherein a bondat the left end bonds to the benzene ring of formula I above and a bondat the right end bonds to the nitrogen heteroatom of formula I above);

R¹⁵ is represented by formula II:

wherein:

R⁶-R⁹ are each independently H, hydroxyl, alkyl, or alkoxy;

R′°—R¹⁴ are each independently H, hydroxyl, alkyl, alkoxy, oraminocarbonyl; and

X is O, S, or N(H).

In certain embodiments, a compound having a structure of

is not included in formula I.

In certain embodiments, R¹-R⁴ are each independently H, hydroxyl oracetamido. According to preferred examples, each of R¹-R⁴ is H; one ofR¹-R⁴ is acetamido (preferably R³) and the remaining R¹-R⁴ are each H;or one of R¹-R⁴ is hydroxyl (preferably R⁴) and the remaining R¹-R⁴ areeach H.

In certain embodiments, R⁵ is an aralkyl such as —C₁-C₄ alkyl —Ar(wherein Ar is a 6-member or 5-member ring). According to preferredexamples, R⁵ is —CH₂-Ph (wherein Ph is a phenyl or substituted phenylgroup); or —CH₂-heteroAr (wherein heteroAr is an aryl ring that includesat least one heteroatom such as O, N or S (e.g., a furyl group).

In certain embodiments, A represents —N═C(R¹⁵)— (wherein a bond at theleft end bonds to the benzene ring of formula I above and a bond at theright end bonds to the nitrogen heteroatom of formula I above) resultingin a structure of formula IV:

(although not shown in formula V, R¹-R⁴ are also present as shown informula I).

In other embodiments, A represents —NH—CH(R¹⁵)— (wherein a bond at theleft end bonds to the benzene ring of formula I above and a bond at theright end bonds to the nitrogen heteroatom of formula I above) resultingin a structure of formula V;

(although not shown in formula V, R¹-R⁴ are also present as shown informula I).

In certain embodiments, R⁶-R⁸ are each H and R⁹ is alkoxy (particularlymethoxy).

In certain embodiments, R¹⁰, R¹¹, R¹³, and R¹⁴ are each H and R¹² isacetamido.

Preferably, R¹⁵ is represented by formula III:

wherein X is O or S, preferably 0.

Illustrative compounds include:

According to another embodiment, examples of the additional TSHRagonists are represented by formula VI:

wherein R²⁰ is aryl or heteroaryl; R²¹ is aryl or heteroaryl; R²² is R¹⁵of formula II, heteroaryl, or aryl. According to a preferred embodiment,R²¹ is benzyl and R²² is R¹⁵ of formula II (particularly formula III).II. General Synthesis

The compounds disclosed herein may be generally synthesized as describedbelow. With reference to Scheme 1,2-aminobenzamides (1) were prepared byeither amide couplings (step i) of 2-aminobenzoic acids with differentamines or reactions of isatoic anhydrides with amines (step ii).

With reference to Scheme 2, reactions of benzyl chlorides 2 withdifferent phenols or thiophenols under microwave irradiation (step i)generated aldehydes 3. Condensations of aldehydes 3 with2-aminobenzamides 1 yielded 2,3-dihydroquinazolin-4-ones 4. The2,3-dihydroquinazolin-4-ones 4 were rapidly oxidized by DDQ at roomtemperature to produce quinazolin-4-ones 5.

III. Compositions, Administration and Use of the Disclosed Compounds

The compounds disclosed herein may be useful for thyroid cancerscreening, treating thyroid cancer, treating nodular goiter, TSHstimulation to enhance PET scanning and chemotherapy treatment,differential diagnosis of congenital hypothyroidism, treatingosteoporosis (e.g., inhibiting bone loss) and treating overweight orobesity (e.g, increase metabolic rate of fat tissue). Illustrative usesof the compounds disclosed herein include:

agonist thyroglobulin (Tg) testing may be used in patients with anundetectable Tg on thyroid suppression therapy to exclude the diagnosisof residual or recurrent thyroid cancer;

agonist treatment may be used in combination with radioiodine (131I) toablate thyroid remnants following near-total thyroidectomym in patientswithout evidence of metastatic disease;

agonist testing may be used in patients requiring serum Tg testing andradioiodine imaging who are unwilling to undergo thyroid hormonewithdrawal testing and whose treating physician believes that use of aless sensitive test is justified; or

agonist treatment and testing may be used in patients who are eitherunable to mount an adequate endogenous TSH response to thyroid hormonewithdrawal or in whom withdrawal is medially contraindicated.

According to one embodiment the compounds disclosed herein (includingCompound I) are useful for thyroid cancer screening. Radioiodine is usedfor detection of thyroid cancer cells and to increase test sensitivitythe uptake of radioiodine must be enhanced. Administration of a TSHRagonist disclosed herein can increase the iodine uptake of thyroidcells. For example, the TSHR agonists disclosed herein could replacerecombinant human TSH (rhTSH, Thyrogen®, Genzyme) for clinicallyscreening for residual or recurring thyroid cancer after surgery (e.g.,thyroidectomy) in patients with well-differentiated thyroid cancer. TheTSHR agonists disclosed herein also can be used as an adjunct diagnosticfor serum thyroglobulin (TG) testing.

Also disclosed are methods for treating or preventing bone degenerativedisorders that include administering a therapeutically effective amountof at least one TSHR agonist compound disclosed herein. The disorderstreated or prevented include, for example, osteopenia, osteomalacia,osteoporosis, osteomyeloma, osteodystrophy, Paget's disease,osteogenesis imperfecta, bone sclerosis, aplastic bone disorder, humoralhypercalcemic myeloma, multiple myeloma and bone thinning followingmetastasis. The disorders treated or prevented further include bonedegenerative disorders associated with hypercalcemia, chronic renaldisease (including end-stage renal disease), kidney dialysis, primary orsecondary hyperparathyroidism, and long-term use of corticosteroids.

The compounds disclosed herein may be included in pharmaceuticalcompositions (pharmaceutical compositions include therapeutic,diagnostic and prophylactic formulations), typically combined togetherwith one or more pharmaceutically acceptable vehicles or carriers and,optionally, other therapeutic or diagnostic ingredients (for example, aradioiodine). Such pharmaceutical compositions can be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, intranasal, intrapulmonary, or transdermal delivery, or bytopical delivery to other surfaces. Optionally, the compositions can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-arterial, intra-articular,intraperitoneal, intrathecal, intracerebroventricular, or parenteralroutes. In other alternative embodiments, the compound can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the compound can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the compound. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included. Adjuvants, such as aluminum hydroxide (forexample, Amphogel, Wyeth Laboratories, Madison, N.J.), Freund'sadjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many othersuitable adjuvants well known in the art, can be included in thecompositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The compound can be combined with the base or vehicle according to avariety of methods, and release of the compound can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the compound is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the compound can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compound can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the compound can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the compoundand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, thecompound can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the compound and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

The administration of the compound of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, thecompound is provided in advance of any symptom. The prophylacticadministration of the compound serves to prevent or ameliorate anysubsequent disease process. When provided therapeutically, the compoundis provided at (or shortly after) the onset of a symptom of disease orinfection.

For prophylactic and therapeutic purposes, the compound can beadministered to the subject in a single bolus delivery, via continuousdelivery (for example, continuous transdermal, mucosal or intravenousdelivery) over an extended time period, or in a repeated administrationprotocol (for example, by an hourly, daily or weekly, repeatedadministration protocol). The therapeutically effective dosage of thecompound can be provided as repeated doses within a prolongedprophylaxis or treatment regimen that will yield clinically significantresults to alleviate one or more symptoms or detectable conditionsassociated with a targeted disease or condition as set forth herein.Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of thecompound (for example, amounts that are effective to elicit a desiredimmune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the compound may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the compound will vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the compound for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of a compound and/or other biologically active agent within themethods and formulations of the disclosure is about 0.01 mg/kg bodyweight to about 10 mg/kg body weight, such as about 0.05 mg/kg to about5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, or intranasal delivery versus intravenous or subcutaneousdelivery. Dosage can also be adjusted based on the release rate of theadministered formulation, for example, of an intrapulmonary spray versuspowder, sustained release oral versus injected particulate ortransdermal delivery formulations, and so forth. To achieve the sameserum concentration level, for example, slow-release particles with arelease rate of 5 nanomoles (under standard conditions) would beadministered at about twice the dosage of particles with a release rateof 10 nanomoles.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of diseases and other conditions inmammalian subjects. Kits for diagnostic use are also provided. In oneembodiment, these kits include a container or formulation that containsone or more of the conjugates described herein. In one example, thiscomponent is formulated in a pharmaceutical preparation for delivery toa subject. The conjugate is optionally contained in a bulk dispensingcontainer or unit or multi-unit dosage form. Optional dispensing meanscan be provided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

Examples

General Materials and Methods:

All commercially available reagents and solvents were purchased and usedwithout further purification. All microwave reactions were carried outin a sealed microwave vial equipped with a magnetic stir bar and heatedin a Biotage Initiator Microwave Synthesizer. All compounds forbiological testing were purified using a Waters semi-preparative HPLCequipped with a Phenomenex Luna® C18 reverse phase (5 micron, 30×75 mm)column having a flow rate of 45 mL/min. The mobile phase was a mixtureof acetonitrile and H₂O each containing 0.1% trifluoroacetic acid.During purification, a gradient of 30% to 80% acetonitrile over 8minutes was used with fraction collection triggered by UV detection (220nM). Pure fractions passed through PL-HCO₃ MP SPE (Varian) to removetrifluoroacetic acid and concentrated under vacuum on a lyophilizer. ¹Hspectra were recorded using an Inova 400 (100) MHz spectrometer(Varian). Chemical shifts are reported in 6 (ppm) units using ¹H(residual) from CDCl₃ (7.27) as internal standard. Data are reported asfollows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet, br=broad), coupling constant, and integration.Samples were analyzed for purity on an Agilent 1200 series LC/MSequipped with a Zorbax™ Eclipse XDB-C18 reverse phase (5 micron, 4.6×150mm) column having a flow rate of 1.1 mL/min. The mobile phase was amixture of acetonitrile and H₂O each containing 0.05% trifluoroaceticacid. A gradient of 5% to 100% acetonitrile over 8 minutes was usedduring analytical analysis. High-resolution mass spectroscopymeasurements were performed on a Agilent 6210 Electrospray TOF massspectrometer.

General Synthetic Procedures

The following general procedures were used to synthesize compoundshaving different but analogous structures. One of skill in the art willrecognize how to modify these general procedures if necessary toaccomplish the desired transformations.

General Procedure for the Synthesis of 2-Aminobenzamides from IsatoicAnhydride:

To a solution of isatoic anhydride (0.163 g, 1.0 mmol, 1.0 equiv) in 10mL of anhydrous acetonitrile was added amines (1.05 mmol, 1.05 equiv) atroom temperature. The resulting mixture was stirred at room temperaturefor 2 hours and heated at 50° C. for 4 hours. Then, it was concentratedin vacuo yielded the products as solids in 90-99% yields

2-Amino-N-benzylbenzamide

¹H NMR (400 MHz, CHLOROFORM-d) δ 4.61 (s, 1H), 4.63 (s, 1H), 5.58 (br.s., 2H), 6.33 (br. s., 1H), 6.62-6.66 (m, 1H), 6.69-6.71 (m, 1H),7.19-7.25 (m, 1H), 7.28-7.43 (m, 6H); LCMS: (electrospray+ve), m/z 227.1(MH)⁺; HPLC: t_(R)=4.38 min, UV₂₅₄=96%.

2-Amino-N-(furan-2-ylmethyl)benzamide

¹H NMR (400 MHz, CHLOROFORM-d) δ 4.60 (s, 1H), 4.61 (s, 1H), 5.57 (br.s., 2H), 6.24-6.42 (m, 3H), 6.59-6.74 (m, 2H), 7.16-7.25 (m, 1H),7.33-7.39 (m, 2H); LCMS: (electrospray+ve), m/z 217.1 (MH)⁺; HPLC:t_(R)=3.77 min, UV₂₅₄=98%.

2-Amino-N-benzyl-6-methoxybenzamide

To a solution of 2-amino-6-methoxybenzoic acid (0.841 g, 5.0 mmol, 1.0equiv), benzylamine (0.643 g, 6.0 mmol, 1.2 equiv), anddiisopropylethylamine (1.935 g, 15.0 mmol, 3.0 equiv) in 50 mL ofdichloromethane was added 2-chloro-1,3-dimethylimidazolinium chloride(1.099 g, 6.5 mmol, 1.3 equiv) at room temperature. The mixture wasstirred at room temperature for 6 hours, poured into water, andextracted with dichloromethane. The organic solution was successivelywashed with aqueous saturated NaHCO₃ and water. The organic layer wasdried over MgSO₄ and the solvent was removed by rotary evaporator. Theresidue was purified by column chromatography (silica gel, 2% 2.0 Mammonia MeOH solution in CH₂Cl₂) to give2-Amino-N-benzyl-6-methoxybenzamide (0.593 g, 46%) as a solid. ¹H NMR(400 MHz, CHLOROFORM-d) δ 3.81 (s, 3H), 4.62 (s, 1H), 4.63 (s, 1H), 6.07(vb.s, 2H), 6.19 (d, J=8.2 Hz, 1H), 6.32 (d, J=8.2 Hz, 1H), 7.07 (t,J=8.2 Hz, 1H), 7.14-7.54 (m, 5H), 8.05 (br. s., 1H); HPLC: t_(R)=4.72min, UV₂₅₄=99%; HRMS (ESI): m/z calcd for C₁₅H₁₆N₂O₂ [M+1]⁺257.1296.found 257.1294.

2-Amino-N-benzyl-6-hydroxybenzamide

To a solution of 2-amino-N-benzyl-6-methoxybenzamide (0.228 g, 0.89mmol, 1.0 equiv) in 3 mL of anhydrous DMF was added 1-dodecanethiol(0.360 g, 1.78 mmol, 2.0 equiv), followed by adding NaOMe (0.385 g of25% solution in MeOH, 1.78, 2.0 equiv). The mixture was heated in amicrowave at 150° C. for 10 min. The mixture passed through a silica gelplug, which was washed with an ethyl acetate-methanol mixture (1:1). Thecrude product was purified by PL-SO₃H MP SPE (Varian) to give2-Amino-N-benzyl-6-hydroxybenzamide (0.176 g, 82%) as thick oil. ¹H NMR(400 MHz, CHLOROFORM-d) δ 3.55-4.20 (vb.s., 2H), 4.60 (s, 1H), 4.61 (s,1H), 6.25 (d, J=7.8 Hz, 1H), 6.50 (d, J=8.2 Hz, 1H), 7.09 (t, J=8.0 Hz,1H), 7.16-7.52 (m, 5H), 8.58 (br. s., 1H), 12.05 (br. s., 1H); HPLC:t_(R)=3.97 min, UV₂₅₄=98%; HRMS (ESI): m/z calcd for C₁₄H₁₄N₂O₂ [M+1]⁺243.11. found 243.1134.

General procedure for the syntheses of 2,3-dihydroquinazolin-4-ones andquinazolin-4-ones

To a solution of 3-(chloromethyl)-4-methoxybenzaldehyde (300 umol, 1.0equiv) and the appropriately substituted phenol or thiolphenol (360umol, 1.2 equiv) in 1.5 mL of anhydrous DMA was added K₂CO₃ (1.5 mmol, 5equiv). The mixture was heated in a microwave at 150° C. for 10 min. Thesolid was filtered. To the clear solution was added 2-aminobenzamides(1.2 equiv), followed by Ytterbium trifluoromethanesulfonate (150 umol,0.5 equiv). The mixture was heated in a microwave at 200° C. for 10 mingiving the desired 2,3-dihydroquinazolin-4-ones. Adding a DDQ (300 umol,1.5 M in acetonitrile, 1.0 equiv) solution to the reaction mixturecontaining 2,3-dihydroquinazolin-4-ones and stirring the resultedmixture for 1 hour gave quinazolin-4-ones. All crude products werepurified by HPLC. PL-HCO₃ MP SPE was used to remove TFA. The finalproducts were obtained as solids in 20-50% yields.

N-(4-(5-(3-(Furan-2-ylmethyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide(Compound 3)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.11 (s, 3H), 3.72 (d, J=15.6 Hz, 1H),3.83 (s, 3H), 4.25 (v.b.s, 1H), 5.01 (s, 2H), 5.26 (d, J=15.6 Hz, 1H),5.71 (s, 1H), 6.03-6.29 (m, 2H), 6.48 (d, J=7.8 Hz, 1H), 6.83 (dd,J=18.2, 8.8 Hz, 3H), 7.10-7.61 (m, 7H), 7.92 (d, J=7.0 Hz, 1H); HPLC:t_(R)=5.40 min, UV₂₅₄=91%; HRMS (ESI): m/z calcd for C₂₉H₂₇N₃O₅ [M+1]⁺498.2029. found 498.2025.

N-(4-(5-(3-Benzyl-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide(Compound 3/2)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.12 (s, 3H), 3.66 (d, J=15.3 Hz, 1H),3.83 (s, 3H), 5.00 (s, 2H), 5.48 (d, J=15.3 Hz, 1H), 5.57 (s, 1H), 6.49(d, J=7.8 Hz, 1H), 6.67-7.00 (m, 4H), 7.05-7.57 (m, 11H), 7.99 (d, J=7.8Hz, 1H); HPLC: t_(R)=5.69 min, UV₂₅₄=98%; HRMS (ESI): m/z calcd forC₃₁H₂₉N₃O₄ [M+1]⁺ 508.2242. found 508.2233.

N-(4-(5-(3-Benzyl-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzylthio)phenyl)acetamide(Compound 3/5)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.14 (s, 3H), 3.50 (d, J=15.4 Hz, 1H),3.83 (s, 3H), 3.88 (d, J=13.1 Hz, 1H), 4.00 (d, J=13.1 Hz, 1H), 5.42 (d,J=15.3 Hz, 1H), 5.45 (s, 1H), 6.52 (d, J=8.2 Hz, 1H), 6.74 (d, J=8.6 Hz,1H), 6.79 (d, J=2.0 Hz, 1H), 6.84 (t, J=7.4 Hz, 1H), 7.06 (dd, J=8.4,2.2 Hz, 1H), 7.10-7.33 (m, 9H), 7.37 (d, J=8.6 Hz, 2H), 7.68 (br. s.,1H), 7.96 (d, J=6.6 Hz, 1H); HPLC: t_(R)=5.97 min, UV₂₅₄=98%; HRMS(ESI): m/z calcd for C₃₁H₂₉N₃O₃S [M+1]⁺ 524.2002. found 524.2002.

N-(4-(5-(3-Benzyl-5-hydroxy-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide(Compound 3/4)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.16 (s, 3H), 3.67 (d, J=15.5 Hz, 1H),3.87 (s, 3H), 4.38 (bs, 1H), 5.05 (s, 2H), 5.37 (d, J=15.2 Hz, 1H), 5.58(d, J=1.6 Hz, 1H), 5.95 (dd, J=8.0, 1.0 Hz, 1H), 6.35 (dd, J=8.4, 0.9Hz, 1H), 6.80-6.92 (m, 3H), 7.09-7.40 (m, 10H), 12.34 (s., 1H); HPLC:t_(R)=6.11 min, UV₂₅₄=96%; HRMS (ESI): m/z calcd for C₃₁H₂₉N₃O₅ [M+1]⁺524.2185. found 524.2184.

N-(4-(5-(3-(Furan-2-ylmethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide(Compound 3/1)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.15 (s, 3H), 3.95 (s, 3H), 5.12 (s,2H), 5.19 (s, 2H), 6.12 (d, J=2.7 Hz, 1H), 6.22-6.28 (m, 1H), 6.93 (d,J=9.0 Hz, 2H), 7.00 (d, J=8.6 Hz, 1H), 7.12 (br. s., 1H), 7.24 (s, 1H),7.39 (d, J=9.0 Hz, 2H), 7.46-7.56 (m, 2H), 7.64-7.82 (m, 3H), 8.33 (d,J=8.2 Hz, 1H); HPLC: t_(R)=5.49 min, UV₂₅₄=95%; HRMS (ESI): m/z calcdfor C₂₉H₂₅N₃O₅ [M+1]⁺ 496.1872. found 496.1873.

N-(4-(5-(3-Benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide(Compound 3/3)

¹H NMR (400 MHz, CHLOROFORM-d) δ 2.13 (s, 3H), 3.91 (s, 3H), 5.04 (s,2H), 5.26 (s, 2H), 6.81-7.04 (m, 5H), 7.11-7.26 (m, 4H), 7.38 (d, J=9.0Hz, 2H), 7.46-7.58 (m, 2H), 7.71-7.83 (m, 2H), 8.36 (d, J=7.8 Hz, 1H);HPLC: t_(R)=5.73 min, UV₂₅₄=98%; HRMS (ESI): m/z calcd for C₃₁H₂₇N₃O₄[M+1]⁺ 506.2086. found 506.2082.

Generation of Stable Cell-Lines Expressing TSHR, LHCGR or FSHR.

cDNA for human TSHR was amplified by PCR from hTSHR-pSVL (1) andinserted into the pcDNA3.1(−)/hygromycin vector using restriction sitesXhoI and BamHI. cDNA for human LHCGR was amplified by PCR from hLHR-pGS5(2) and was inserted into the pcDNA3.1(+)/hygromycin vector usingrestriction sites BamHI and XhoI. The FSHR cDNA in pcDNA3.1 was obtainedfrom the Missouri S&T cDNA Resource Center (www.cDNA.org) and wassubcloned into the pcDNA3.1(−)/hygromycin vector. Constructs wereconfirmed by sequencing (MWG Biotech).

HEK-EM 293 cells were transfected with the cDNA of TSHR, LHCGR or FSHRusing FuGENE 6 Transfection reagent (Roche Diagnostics) according to themanufacturer's protocol. Two days after transfection cells were passagedand grown in Dulbecco's modified Eagle's Medium (DMEM) supplemented with10% fetal bovine serum, 100 units/ml penicillin and 10 μg/mlstreptomycin (Life Technologies Inc.) and with hygromycin (250 μg/ml) asa selection marker. After 7 to 10 days hygromycin-resistant clones wereselected, and after a few days of further growth submitted for a cAMPassay to identify clones that stably express the appropriate receptor.

Cell Culture.

Cells stably expressing TSHR, LHCGR or FSHR and parental HEK 293 cellswere maintained in DMEM medium containing 10% FBS, 100 units/mlPenicillin, 100 μg/ml Streptomycin, at 37° C. in 5% CO₂. For the cellslines stably expressing TSHR, LHCGR or FSHR, additional 250 μg/mlHygromycin was added during the cell culture. The cells were seeded at adensity of 3 to 4 million cells in a T175 flask containing 35 ml ofmedia and were allowed to grow for 3 days to reach 80-90% confluence. Aflask of HEK 293 cells at this density generally yielded 30 millioncells total.

Homogeneous Time Resolved Fluorescence (HTRF) cAMP Assay.

Compounds were assayed using a HTRF cAMP detection kit (Cisbio, Bedford,Mass.) on both TSHR cell line and parental cell line. Briefly, 750 cellswere plated in 2.5 μl/well of complete media (DMEM containing 10% FCS)in 1536 well solid bottom white plates and 20 nl/well compound in DMSOsolution or controls was added. Following 30 minute incubation at roomtemperature, 2.5 μl/well of labeled d2 cAMP and 2.5 μl/well of anti-cAMPantibody (both diluted 1:20 in lysis buffer) were added to each wellusing a flying reagent dispenser (Aurora Discovery, San Diego). Plateswere measured using the Envision plate reader (PerkinElmer, Boston,Mass.) with excitation at 330 nm and emissions of 615 nm and 660 nm.

Compound Preparation.

Compounds were serially diluted 1:5 or 1:2.236 in DMSO in 384-wellplates to yield seven or fifteen concentrations (minimally 10 mM, 2 mM,0.4 mM, 80 μM, 16 μM, 3.2 μM and 0.64 μM) and formatted into 1,536-wellplates at 7 μl/well. Final compound concentrations during cellincubation ranged from 1.60 nM to 25.0 μM.

Data Analysis.

The maximal response (100% activity) was determined by the response of30 mU/ml TSH and the basal response (0% activity) was measured by theDMSO control in the TSH screen. The EC₅₀ values of compounds werecalculated from the concentration-response curves by nonlinearregression analysis using Prism software (GraphPad Software, San Diego,Calif.).

Confirmatory cAMP Assay and Test for Selectivity Towards TSHR

50,000 cells/well were plated in complete media (DMEM containing 10%FCS) in 96 well plates. Cells were cultured for 24 h before incubationfor 1 h in serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine(IBMX) (SIGMA) and bovine TSH (1.8 μM) or human LH or FSH (1000 ng/ml)or compounds (0.01 μM-100 μM) in a humidified 5% CO₂ incubator at 37 C.Following aspiration of the medium after incubation with compounds,cells were lysed using lysis buffer of the cAMP-Screen Direct System(Applied Biosystems, Cat #CSD200). The cAMP content of the cell lysatewas determined using the manufacturer's protocol. The potency (EC₅₀) wasobtained from dose response curves (0-100 μM compound) by data analysiswith GraphPad Prism 4 for Windows.

Below is a List of Compounds that were Assayed Via the Homogeneous TimeResolved Fluorescence (HTRF) cAMP Assay.

Maximum Activity AC50 Log compared Structure (μM) AC50 to TSH

12.59 −4.90 159.24

11.22 −4.95 217.29

 4.47 −5.35  93.95

 7.08 −5.15 171.91

15.85 −4.80 174.77

17.78 −4.75 180.81

 8.91 −5.05 152.47

14.13 −4.85 115.96

17.78 −4.75 115.62

 7.94 −5.10 122.44

 3.16 −5.50 113.94

 3.98 −5.40  91.43

 2.24 −5.65 103.29

11.22 −4.95 104.66

12.59 −4.90 123.21

19.95 −4.70  97.95

10.00 −5.00  93.46

 6.31 −5.20 215.89

15.85 −4.80 142.18

14.13 −4.85 156.28

25.12 −4.60 236.35

 7.94 −5.10 463.89

15.85 −4.80 165.50

 7.08 −5.15 159.76

 8.91 −5.05 207.56

10.00 −5.00 217.05

 4.47 −5.35 123.52

10.00 −5.00 405.10

17.78 −4.75 138.99

 3.16 −5.50  63.65

11.22 −4.95 153.40

15.85 −4.80 137.62

17.78 −4.75 168.66

15.85 −4.80 122.30

12.59 −4.90 115.01

 5.62 −5.25 115.47

 8.91 −5.05 104.22

 7.08 −5.15 573.36

19.95 −4.70 123.60

11.22 −4.95 128.80

15.85 −4.80 122.50

14.13 −4.85 131.51

10.00 −5.00 128.45

 7.08 −5.15 105.58

14.13 −4.85 141.85

 7.94 −5.10 182.52

25.12 −4.60 165.16

 8.91 −5.05 106.48

 3.55 −5.45 134.24

 2.82 −5.55  70.37

15.85 −4.80 174.87

 8.91 −5.05 104.78

12.59 −4.90 141.76

10.00 −5.00 115.69

 7.94 −5.10 194.91

11.22 −4.95 110.90

14.13 −4.85 100.67

 8.91 −5.05  93.09

17.78 −4.75 226.43

 6.31 −5.20 189.97

 7.94 −5.10 302.11

 7.08 −5.15 241.92

 5.01 −5.30 166.46

 7.94 −5.10  70.53

14.13 −4.85  51.63

 8.91 −5.05  64.52

 7.94 −5.10  82.36

22.39 −4.65  74.79

25.12 −4.60  44.83

10.00 −5.00  53.96

 7.94 −5.10  44.19

 3.98 −5.40  55.25

28.18 −4.55  75.04

 2.82 −5.55  36.00

19.95 −4.70  70.09

 6.31 −5.20  64.77

 3.16 −5.50  68.63

 8.91 −5.05  66.16

 1.78 −5.75  61.52

22.39 −4.65  40.00

15.85 −4.80  76.98

25.12 −4.60  58.96

 7.08 −5.15  79.03

 7.94 −5.10  47.53

12.59 −4.90  54.95

 6.31 −5.20  51.11

 3.98 −5.40  38.00

 5.62 −5.25  30.50

25.12 −4.60 129.12

44.67 −4.35 110.89

15.85 −4.80  88.07

31.62 −4.50 133.48

28.18 −4.55 111.66

25.12 −4.60 160.25

12.59 −4.90 138.18

14.13 −4.85 124.86

15.85 −4.80 132.18

14.13 −4.85 140.53

35.48 −4.45 104.15

35.48 −4.45 190.35

28.18 −4.55 128.51

15.85 −4.80 88.66

15.85 −4.80 106.72

39.81 −4.40 122.31

17.78 −4.75 125.72

17.78 −4.75  45.41

31.62 −4.50  63.73

15.85 −4.80  63.12

17.78 −4.75  47.53

22.39 −4.65  48.00

35.48 −4.45  98.46

15.85 −4.80  60.92

39.81 −4.40  63.82

25.12 −4.60  51.15

39.81 −4.40  66.81

35.48 −4.45  54.57

44.67 −4.35  87.19

28.18 −4.55  96.91

14.13 −4.85  56.31

15.85 −4.80  40.50

39.81 −4.40  38.00

50.12 −4.30  61.90

14.13 −4.85  83.18

31.62 −4.50  62.73

39.81 −4.40  56.43

17.78 −4.75  36.00

14.13 −4.85  38.00

39.81 −4.40  69.13

28.18 −4.55  51.77

50.12 −4.30  53.70

35.48 −4.45  99.01

31.62 −4.50  44.00

31.62 −4.50  55.28

 8.91 −5.05  30.50

44.67 −4.35  48.07

28.18 −4.55  37.00

inactive

19.95 −4.70  31.50

inactive

inactive

inactive

19.95 −4.70  34.00

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

12.59 −4.90  25.50

inactive

inactive

inactive

inactive

50.12 −4.30  28.00

inactive

inactive

inactive

inactive

inactive

inactive

14.13 −4.85  24.50

inactive

50.12 −4.30  34.00

inactive

inactive

inactive

15.85 −4.80  30.00

inactive

inactive

inactive

 5.62 −5.25  29.00

inactive

inactive

inactive

 3.98 −5.40  22.50

inactive

inactive

inactive

inactive

inactive

inactive

inactive

22.39 −4.65  24.00

inactive

inactive

inactive

inactive

31.62 −4.50  27.50

inactive

inactive

 2.82 −5.55  17.00

19.95 −4.70  18.00

inactive

28.18 −4.55  38.00

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

 5.62 −5.25  20.00

inactive

inactive

39.81 −4.40  38.00

inactive

17.78 −4.75  32.00

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

 8.91 −5.05  23.50

inactive

 3.55 −5.45  16.00

inactive

inactive

 8.91 −5.05  18.00

inactive

35.48 −4.45  32.00

inactive

inactive

17.78 −4.75  30.00

inactive

inactive

inactive

inactive

inactive

inactive

inactive

inactive

70.79 −4.15  32.00

50.12 −4.30  38.00

inactive

inactive

inactive

inactive

inactive

inactive

 5.01 −5.30  24.00

19.95 −4.70  20.00

28.18 −4.55  21.50

inactive

inactive

inactive

inactive

inactive

inactive

 6.31 −5.20  24.00

inactive

 7.94 −5.10  24.50

inactive

25.12 −4.60  28.00

inactive

inactive

inactive

 2.51 −5.60  11.00

19.95 −4.70  28.00

 7.94 −5.10  23.50

25.12 −4.60  32.00

inactive

inactive

 4.47 −5.35  20.50

17.78 −4.75  24.00

 5.62 −5.25  22.00

inactive

inactive

inactive

inactive

inactive

22.39 −4.65  30.00

 2.24 −5.65  20.50

inactive

inactive

35.48 −4.45  38.00

inactive

inactive

inactiveThe EC₅₀ Values Determined Via the Confirmatory cAMP Assay for SeveralCompounds were:

Compound 3 590 nM Compound 3/4  25 nM Compound 3/5  38 nM Compound 3/1100 nM Compound 3/2 541 nM

FIG. 1 shows that the compounds disclosed herein are TSHR-selectiveagonists since they exhibit no agonist activity against LHCGR or FSHR.

FIG. 2 is a graph depicting data in HEK EM 293 cells stably expressingthe TSHR demonstrating the potency of several compounds disclosedherein.

FIG. 3 is a graph of data for the agonist activity of several compoundsdisclosed herein in primary cultures of human thyrocytes.

Because we identified the compounds disclosed herein using HEK EM 293cells stably expressing TSHR, we sought to confirm their activities inprimary cultures of human thyrocytes. Since TSH up-regulates expressionof thyroid specific genes, we tested compounds disclosed herein onexpression of mRNAs for thyroglobulin (TG) and thyroperoxidase (TP0).After 24 h, treatment of thyrocytes with 30 uM compounds disclosedherein increased TG mRNA expression to a level similar to TSH whereasTSHR small agonists increased TP0 mRNA expression but to lower levelsthan bTSH. This demonstrates that these ligands are active in primarycultures of human thyrocytes that express TSHR at physiological levels.

Culture of Primary Human Thyrocytes

Thyroid tissue samples were obtained through the National Institutes ofHealth Clinical Center during surgery for unrelated reasons. Patientsprovided informed consent on an IRB approved protocol and materials werereceived anonymously via approval of research activity through theOffice of Human Subjects Research. The specimens were maintained in HBSSon ice and isolation of cells was initiated within 4 h after surgery.All preparations were performed under sterile conditions. Tissue sampleswere minced into small pieces by fine surgical forceps and scissors in a10 cm dish with a small volume of HBSS. Tissue pieces were transferredto a 15 ml tube (Falcon) and washed at least 3 times with HBSS.Afterwards, tissue pieces were incubated with HBSS containing 3 mg/mlCollagenase Type IV (Gibco). Enzymatic digestion proceeded for 30 min orlonger with constant shaking in a water bath at 37° C. until asuspension of isolated cells was obtained. After centrifugation for 5min at 1000 rpm, the supernatant was removed and cells were resuspendedin 10 ml DMEM with 10% FBS. Cells were plated in 10 cm tissue culturedishes and incubated at 37° C. in a humidified 5% CO₂ incubator. After24 h, the supernatant containing non-adherent cells was removed. Theprimary cultures of thyroid cells formed a confluent monolayer within5-7 days. For determination of thyroglobulin and thyroperoxidase mRNAexpression, thyrocytes were seeded into 24-well plates at a density of6×10⁴ cells/well 24 h before the experiment.

Quantitative RT-PCR

Total RNA was purified using RNeasy Micro kits (Qiagen). First strandcDNA was prepared using a High Capacity cDNA Archive Kit (AppliedBiosystems). RT-PCR was performed in 25 μl reactions using cDNA preparedfrom 100 ng of total RNA and Universal PCR Master Mix (AppliedBiosystems). Primers and probes for thyroglobulin and thyroperoxidasewere Assay-on-Demand (Applied Biosystems). Quantitative RT-PCR resultswere normalized to GAPDH to correct for differences in RNA input.

FIG. 4 is a graph of data of in vivo activity of a compound disclosedherein in mice

We have shown in vitro using transfected cells that compounds disclosedherein activate the mouse TSHR with similar potency and efficacy ashuman TSHR. Because the known physiology of TSHRs is similar in mice andhumans, in vivo studies in mice offer an ideal preclinical model forassessing possible clinical applications for these compound disclosedherein. We determined if one compound disclosed herein can activate TSHRwhen administered by intraperitoneal injection. 20 ug of the compounddisclosed herein and 30 ug TSH in PBS containing 2% DMSO wereadministered to unanesthetized C57 BL/6 mice. The experimental endpointwas total thyroxine (T4) measurement in serum obtained from terminalretroorbital bleeds from anesthetized mice 2 hours after administrationof the compound or TSH. Total T4 levels did rise after injection of TSHand of the compound disclosed herein.

In view of the many possible embodiments to which the principles of thedisclosed compounds, compositions and methods may be applied, it shouldbe recognized that the illustrated embodiments are only preferredexamples and should not be taken as limiting the scope of the invention.Rather, the scope of the invention is defined by the following claims.We therefore claim as our invention all that comes within the scope andspirit of these claims.

What is claimed is:
 1. A compound having formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹-R⁴ are eachindependently H, hydroxyl, alkyl, alkoxy, aminocarbonyl, or halogen; R⁵is —CH₂-Ph, wherein Ph is a substituted phenyl; A represents —N═C(R¹⁵)—(wherein a bond at the left end bonds to the benzene ring of formula Iabove and a bond at the right end bonds to the nitrogen heteroatom offormula I above) or —NH—CH(R¹⁵)— (wherein a bond at the left end bondsto the benzene ring of formula I above and a bond at the right end bondsto the nitrogen heteroatom of formula I above); R¹⁵ is represented byformula II:

wherein: R⁶-R⁹ are each independently H, hydroxyl, alkyl, or alkoxy;R¹⁰, R¹¹, R¹³ and R¹⁴ are each independently H; R¹² is acetamido; and Xis O, S or N(H).
 2. The compound of claim 1, wherein R⁹ is C₁-C₄ isalkoxy.
 3. The compound of claim 1, wherein X is O.
 4. The compound ofclaim 1, wherein R¹-R⁴ are each independently H.
 5. The compound ofclaim 1, wherein R¹⁵ is represented by formula III:


6. The compound oaf claim 1, wherein the compound is a thyroidstimulating hormone receptor agonist.
 7. The compound of claim 1,wherein the compound is a selective thyroid stimulating hormone receptoragonist.
 8. The compound of claim 1, wherein R⁹ methoxy; and X is O orS.
 9. A method for detecting thyroid cancer in a subject, comprisingadministering to the subject in need thereof a diagnostically effectiveamount of a compound of claim 1 and a diagnostically effective amount ofradioiodine.
 10. The method of claim 9, wherein the subject hasundergone thyroidectomy for well-differentiated thyroid cancer.
 11. Themethod of claim 9, wherein administration of the at least one compoundof formula I enhances iodine uptake by thyroid cells.
 12. A method fortreating thyroid cancer in a subject, comprising administering to thesubject in need thereof a therapeutically effective amount of a compoundof claim
 1. 13. The method of claim 12, further comprisingco-administering radioiodine with the compound of formula I to ablatethyroid remnants following near-total thyroidectomy in a subject withoutevidence of metastatic disease.
 14. The compound of claim 1, wherein R⁵is —CH₂-Ph wherein Ph is a phenyl substituted with chloro, phenyl,methoxy, trifluoromethyl, or methyl.
 15. The compound of claim 5,wherein R⁵ is —CH₂-Ph, wherein Ph is a phenyl substituted with chloro,phenyl, methoxy, trifluoromethyl, or methyl.
 16. The compound of claim1, wherein R⁵ is —CH₂-Ph, wherein Ph is a phenyl substituted with3-chloro.
 17. The compound of claim 1, wherein R⁹ is methoxy; X is O; Ais —NH—CH(R¹⁵)—; and R¹-R⁴ and R⁷-R⁸ are each independently H.
 18. Thecompound of claim 17, wherein R⁵ is —CH₂-Ph, wherein Ph is a phenylsubstituted with 3-chloro.
 19. The method of claim 9, wherein theradioiodine is radioiodine I-131.
 20. The method of claim 9, wherein themethod further comprises measuring serum thyroglobulin.
 21. The methodof claim 13, wherein the radioiodine is radioiodine I-131.